Carol Holm-Hansen

Comparison of oral fluid collectors for use in a rapid point-of-care diagnostic device.

ABSTRACT Orally based diagnostic testing is emerging as an alternative, noninvasive method for analyzing a variety of analytes. These analytes include pathogens, antibodies, drugs, and nucleic acids. In the present study we developed a protocol for evaluation of collectors that could be used in orally based, point-of-care diagnostics. A performance comparison was carried out with a number of commercially available collectors, and their ability to deliver fluid, proteins, bacteria, and nucleic acid from pathogens compatible with PCR was assessed. The collectors were all capable of picking up and delivering test materials, albeit at various levels.

Mycobacterium tuberculosis Lineage 7 Strains Are Associated with Prolonged Patient Delay in Seeking Treatment for Pulmonary Tuberculosis in Amhara Region, Ethiopia

ABSTRACT Recent genotyping studies of Mycobacterium tuberculosis in Ethiopia have reported the identification of a new phylogenetically distinct M. tuberculosis lineage, lineage 7. We therefore investigated the genetic diversity and association of specific M. tuberculosis lineages with sociodemographic and clinical parameters among pulmonary TB patients in the Amhara Region, Ethiopia. DNA was isolated from M. tuberculosis-positive sputum specimens (n = 240) and analyzed by PCR and 24-locus mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) analysis and spoligotyping. Bioinformatic analysis assigned the M. tuberculosis genotypes to global lineages, and associations between patient characteristics and genotype were evaluated using logistic regression analysis. The study revealed a high diversity of modern and premodern M. tuberculosis lineages, among which approximately 25% were not previously reported. Among the M. tuberculosis strains (n = 138) assigned to seven subgroups, the largest cluster belonged to the lineage Central Asian (CAS) (n = 60; 26.0%), the second largest to lineage 7 (n = 36; 15.6%), and the third largest to the lineage Haarlem (n = 35; 15.2%). Four sublineages were new in the MIRU-VNTRplus database, designated NW-ETH3, NW-ETH1, NW-ETH2, and NW-ETH4, which included 24 (10.4%), 18 (7.8%), 8 (3.5%), and 5 (2.2%) isolates, respectively. Notably, patient delay in seeking treatment was significantly longer among patients infected with lineage 7 strains (Mann-Whitney test, P < 0.008) than in patients infected with CAS strains (adjusted odds ratio [AOR], 4.7; 95% confidence interval [CI], 1.6 to 13.5). Lineage 7 strains also grew more slowly than other M. tuberculosis strains. Cases of Haarlem (OR, 2.8; 95% CI, 1.2 to 6.6) and NW-ETH3 (OR, 2.8; 95% CI, 1.0 to 7.3) infection appeared in defined clusters. Intensified active case finding and contact tracing activities in the study region are needed to expedite diagnosis and treatment of TB.

Primary drug resistance to anti-tuberculosis drugs in major towns of Amhara region, Ethiopia.

Yimer SA, Agonafir M, Derese Y, Sani Y, Bjune GA, Holm‐Hansen C. Primary drug resistance to anti‐tuberculosis drugs in major towns of Amhara Region, Ethiopia. APMIS 2012; 120: 503–9.

Prevalence of reverse transcriptase and protease mutations associated with antiretroviral drug resistance among drug-naïve HIV-1 infected pregnant women in Kagera and Kilimanjaro regions, Tanzania

BackgroundAccess to antiretroviral drugs for HIV-1 infection has increased in sub-Saharan Africa (SSA) during the past few years. Mutations in the HIV-1 genome are often associated with treatment failure as indicated by viral replication and elevated levels of virus in the blood. Mutations conferring resistance to antiretroviral drugs are based on comparing gene sequences with corresponding consensus sequences of HIV-1 subtype B that represents only 10% of the AIDS pandemic. The HIV pandemic in SSA is characterized by high viral genetic diversity. Before antiretroviral drugs become more widely available, it is important to characterize baseline naturally occurring genetic mutations and polymorphisms associated with antiretroviral drug resistance among circulating HIV-1 subtypes.MethodsThe prevalence of mutations associated with antiretroviral drug resistance in protease (PR) and reverse transcriptase (RT) regions among antiretroviral treatment-naïve HIV-1 infected pregnant women was investigated in Bukoba (Kagera) and Moshi (Kilimanjaro) municipalities, Tanzania, between September and December 2005. The HIV-1 pol gene was amplified using primers recognizing conserved viral sequences and sequenced employing BigDye chemistry from 100 HIV-1 seropositive treatment-naïve pregnant women and 61 HIV-1 seropositive women who had received a single dose of Nevirapine (sdNVP). Positions 1–350 of the RT and 1–99 of the PR genes were analyzed for mutations based on the Stanford University HIV Drug Resistance Database.ResultsHIV-1 subtypes A, C, D, CRF10_CD and Unique Recombinant Forms (URF) were detected. Primary mutations associated with NRTI and NNRTI resistance were detected among 3% and 4% of treatment-naïve strains, respectively. Primary mutations associated with NRTI and NNRTI resistance were detected in 1.6% and 11.5% of women who had received sdNVP, respectively. None of the primary mutations associated with PI resistance was found. Polymorphisms detected in RT and PR sequences were mainly mutations that are found in the consensus sequences of non-B subtypesConclusionBased on the WHO HIV Drug Resistance Research Network Threshold of less than 5%, the baseline prevalence of primary mutations among treatment-naïve HIV-1 infected pregnant women in Kagera and Kilimanjaro regions was low. The significance of HIV-1 subtype B polymorphic positions with respect to antiretroviral resistance identified among the prevalent HIV-1 subtypes is unknown. More studies addressing the correlation between polymorphic mutations, antiretroviral resistance and clinical outcome are warranted in regions where non-B subtypes are prevalent.

Diversity of human immunodeficiency virus type 1 subtypes in Kagera and Kilimanjaro regions, Tanzania.

A strategy to prevent the spread of HIV-1 worldwide is complicated by the high genetic diversity of the virus. To gain a better understanding of the HIV-1 genetic diversity in Tanzania, a molecular epidemiological investigation was conducted in Kagera and Kilimanjaro regions. While several studies have addressed HIV-1 subtypes in Tanzania, this is the first study to describe the virus subtypes circulating in Kagera. The Kagera region is the epicenter of the HIV-1 epidemic in Africa, and it was therefore of interest to compare the prevalence of HIV subtypes in this region and Kilimanjaro. Blood samples were obtained from 246 HIV-1-infected pregnant women attending antenatal clinics. Plasma HIV-1 RNA was extracted, amplified, and sequenced in the env C2V3 and/or pol regions from 209 samples. Based on the analysis of env C2V3 and pol sequences, 47.4% had concordant subtypes, 19.1% were discordant indicating recombination, and for 33.5% sequences were obtained for only one region. The distribution HIV-1 subtypes based on the phylogenetic analysis of paired env C2V3/ pol sequences in Kagera region was A/A (27.8%), C/C (29.6%), D/D (16.7%), and unique recombinant forms (25.9%), and in Kilimanjaro region was A/A (32.9%), C/C (25.9%), D/D (10.6%), CRF10_CD (1.2%), and unique recombinant forms (29.4%). The env C2V3 subsubtype A2 and env C2V3/pol CRF10_CD were also observed indicating that these recombinants are circulating in Tanzania. The high diversity of HIV-1 subtypes and the high prevalence of recombinants demonstrated in this study necessitate expanded and continuous monitoring of the epidemic in Tanzania. The trend may have implications for current national control strategies against the HIV-1 epidemic.

Trends in HIV-1 prevalence and risk behaviours over 15 years in a rural population in Kilimanjaro region of Tanzania

BackgroundMonitoring dynamics in HIV-1 infection and risk behaviours is important in evaluating, adjusting and scaling up prevention programmes. The objective of this study was to estimate trends in the prevalence of HIV-1 infection and risk behaviours over 15 years in a rural village population in Kilimanjaro region of Tanzania using repeated population-based cross-sectional surveys.MethodsFour rounds of HIV-1 sero-epidemiological and behavioural surveys were completed during 1991 to 2005 in the study village. House-to-house registrations of people aged 15–44 years with an address in the village were conducted before each survey. All consenting individuals were then interviewed for pertinent risk behaviours and tested for HIV-1 seropositivity.ResultsParticipation proportions ranged from 73.0% to 79.1%. Overall, age and sex-adjusted HIV-1 prevalence increased from 3.2% in 1991 to 5.6 % in 2005 (relative increase 75.0%; ptrend < 0.001). The increase was significant for both men and women (ptrends < 0.001) and more evident among women aged 35–44 years (2.0% to 13.0%, ptrend < 0.001). Among participants aged 15–24 years a decrease in number of sexual partners was observed with a corresponding stable HIV-1 prevalence. Participants aged 25–44 years continued to report multiple sexual partners, and this was corroborated with increased HIV-1 prevalence trend (4.0% to 9.0%, ptrends < 0.001). Among men aged 25–44 years and women aged 15–24 years significant increases in condom use were observed (ptrend < 0.01).ConclusionThe HIV-1 prevalence seems to have increased among older participants but remained stable among younger participants. Encouraging trends toward safer sex practices were observed among young participants, while only modest behavioural changes were seen among the older participants. Prevention efforts in rural areas need to be intensified and to address people of all ages.

Saliva-Based HIV Testing among Secondary School Students in Tanzania using the OraQuick® Rapid HIV1/2 Antibody Assay

Abstract:  HIV prevalence and knowledge concerning HIV prevention among secondary school students in Tanzania was investigated. Approximately 50% of all secondary school students in Hai district and Moshi town were included in the study. Saliva samples were obtained using the OraQuick® rapid HIV‐1/2 antibody assay. Forty‐one (1.0%) and 211 (5.5%) students at the rural and urban schools, respectively, tested positive for HIV antibodies in saliva. HIV knowledge and beliefs varied significantly. Noninvasive saliva sample collection for HIV testing was highly acceptable. HIV infection is considerably more widespread among students attending urban rather than rural schools in the population investigated.

Intranasal Administration of a Therapeutic HIV Vaccine (Vacc-4x) Induces Dose-Dependent Systemic and Mucosal Immune Responses in a Randomized Controlled Trial

Background Vacc-4x, a Gag p24-based therapeutic HIV vaccine, has been shown to reduce viral load set-points after intradermal administration. In this randomized controlled pilot study we investigate intranasal administration of Vacc-4x with Endocine as adjuvant. Methods Safety and immunogenicity were tested in patients on effective ART. They were randomized to low, medium or high dose Vacc-4x or adjuvant alone, administered four times at weekly intervals with no booster. Vacc-4x-specific T cell responses were measured in vitro by proliferation and in vivo by a single DTH skin test at the end of study. Nasal and rectal mucosal secretions were analyzed for Vacc-4x-specific antibodies by ELISA. Immune regulation induced by Vacc-4x was assessed by functional blockade of the regulatory cytokines IL-10 and TGF-β. Results Vacc-4x proliferative T cell responses increased only among the vaccinated (p≤0.031). The low dose group showed the greatest increase in Vacc-4x CD8+T cell responses (p = 0.037) and developed larger DTH (p = 0.005) than the adjuvant group. Rectal (distal) Vacc-4x IgA and IgG antibodies also increased (p = 0.043) in this group. In contrast, the high dose generated higher nasal (local) Vacc-4x IgA (p = 0.028) and serum IgG (p = 0.030) antibodies than the adjuvant. Irrespective of dose, increased Vacc-4x CD4+T cell responses were associated with low proliferation (r = −0.82, p<0.001) and high regulation (r = 0.61, p = 0.010) at baseline. Conclusion Intranasal administration of Vacc-4x with Endocine was safe and induced dose-dependent vaccine-specific T cell responses and both mucosal and systemic humoral responses. The clinical significance of dose, immune regulation and mucosal immunity warrants further investigation. Trial Registration ClinicalTrials.gov NCT01473810

Assessment of measles immunity among infants in Maputo City, Mozambique

BackgroundThe optimum age for measles vaccination varies from country to country and thus a standardized vaccination schedule is controversial. While the increase in measles vaccination coverage has produced significant changes in the epidemiology of infection, vaccination schedules have not been adjusted. Instead, measures to cut wild-type virus transmission through mass vaccination campaigns have been instituted. This study estimates the presence of measles antibodies among six- and nine-month-old children and assesses the current vaccination seroconversion by using a non invasive method in Maputo City, Mozambique.MethodsSix- and nine-month old children and their mothers were screened in a cross-sectional study for measles-specific antibodies in oral fluid. All vaccinated children were invited for a follow-up visit 15 days after immunization to assess seroconversion.Results82.4% of the children lost maternal antibodies by six months. Most children were antibody-positive post-vaccination at nine months, although 30.5 % of nine month old children had antibodies in oral fluid before vaccination. We suggest that these pre-vaccination antibodies are due to contact with wild-type of measles virus. The observed seroconversion rate after vaccination was 84.2%.ConclusionThese data indicate a need to re-evaluate the effectiveness of the measles immunization policy in the current epidemiological scenario.

Spoligotyping of Mycobacterium tuberculosis isolates among pulmonary tuberculosis patients in Amhara Region, Ethiopia

The aim of this study was to characterize Mycobacterium tuberculosis isolates circulating in the Amhara Region of Ethiopia. Sputum samples were collected from new pulmonary tuberculosis (TB) patients in the Region. Genotyping of mycobacterial DNA was performed by spoligotyping and isolates were assigned to families using the SpolDB4 and the model‐based program ‘Spotclust’. A high level of diversity was found among the 237 isolates. Sixty‐five different spoligopatterns were obtained. The T (30.8%), Central Asian (CAS; 21.1%) and U (17.7%) families were the predominant isolates comprising 69.6% of the total strains. Eighty‐five per cent of the U lineage belonged to Spoligo‐International‐Type (SIT) 910 and SIT 1729. Only a few of these strains are included in SpolDB4 to date. Of the total strains, 41 (17.3%) were unique and have not been described in SpolDB4 to date. This study indicated that the TB epidemic in Amhara Region, Ethiopia, is characterized by the circulation of numerous M. tuberculosis strain families. The high proportion of SIT 910 and SIT 1729 strains may indicate an increase in the importance of these lineages in the transmission of TB in the study region.